Calicheamicin and calicheamicin derivatives refer to a family of antibacterial and antitumor agents, as described, for instance, in U.S. Pat. No. 4,970,198 (which is incorporated herein in its entirety). Calicheamicin derivatives within the scope of the disclosure include, without limitation, dihydro derivatives as described in U.S. Pat. No. 5,037,651 and N-acylated derivatives as described in U.S. Pat. No. 5,079,233 (both of which are incorporated in their entirety herein). As used herein, a calicheamicin derivative refers to calicheamicin that has been substituted at one or more positions to obtain a different compound.
The calicheamicin family of antibiotics, and derivatives and analogs thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., (1993) Cancer Research 53:3336-3342; Angew Chem. Intl. Ed. Engl. (1994) 33:183-186; Lode et al., (1998) Cancer Research 58:2925-2928). Calicheamicin comprises a warhead comprising an enediyne ring structure (a ring comprising a double bond flanked by triple bonds) and a methyl trisulfide (i.e., —S—S—S—CH3) group. It is believed that the warhead is activated by reduction of a disulfide bond, and that the activated warhead functions by causing breaks in double-stranded DNA. A mechanism of action was proposed by Bouchard, H., et al., Ab-drug conjugates-A new wave of cancer drugs, Bioorganic & Medicinal Chemistry Letters 24 (2014) 5357-5363 where the enediyne ring is activated by reductive cleavage of the disulfide bond by the steps: (i) formation of a calicheamicin=CHCH2SH moiety by nucleophilic attack of the methyl trisulfide moiety and cleavage of CH3—S—S—, (ii) formation of a fused 2,5-dihydrothiophene ring from calicheamicin=CHCH2SH, and (iii) formation of a fused benzene free di-radical from the enediyne. Activated calicheamicin then cleaves double stranded DNA.
Calicheamicin has intracellular sites of action, but, in some instances, does not effectively cross the plasma membrane. Therefore, cellular uptake of these agents through antibody-mediated internalization may, in some embodiments, greatly enhance cytotoxic effect. It is known that calicheamicin-linker-antibody conjugates provide for the specificity and effective plasma membrane permeability (internalization) of the antibody in combination with the cytotoxic potency of calicheamicin. Therefore, cellular uptake of calicheamicin may, in some aspects, greatly enhance its cytotoxic effect. Methods of forming calicheamicin-linker-antibody drug conjugates are known and described, for example, in U.S. Pat. Nos. 5,877,296, 5,773,001, 5,712,374, 5,714,586, 5,739,116 and 5,767,285 (each of which is incorporated by reference herein).
Antibody-drug conjugates, comprising an antibody-linker-drug conjugate, are attractive targeted chemo-therapeutic molecules, as they combine ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to the antigen-expressing tumor cells, thereby enhancing their anti-tumor activity. Successful antibody-drug conjugate development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency and mode of linker-drug conjugation to the antibody. More particularly, effective antibody-drug conjugates are characterized by at least one or more of the following: (i) an antibody-drug conjugate formation method wherein the antibody retains sufficient specificity to target antigens and wherein the drug efficacy is maintained; (ii) antibody-drug conjugate stability sufficient to limit drug release in the blood and concomitant damage to non-targeted cells; (iii) sufficient cell membrane transport efficiency (endocytosis) to achieve a therapeutic intracellular antibody-drug conjugate concentration; (iv) sufficient intracellular drug release from the antibody-drug conjugate sufficient to achieve a therapeutic drug concentration; and (v) drug cytotoxicity in nanomolar or sub-nanomolar amounts.
Conventional means of attaching, i.e., covalent bonding of a drug moiety to an antibody via a linker, generally leads to a heterogeneous mixture of molecules where the drug moieties are attached at a number of sites on the antibody. For example, cytotoxic drugs have typically been conjugated to antibodies through the often-numerous lysine residues of an antibody, generating a heterogeneous antibody-drug conjugate mixture. Depending on reaction conditions, the heterogeneous mixture typically contains a distribution of antibodies with from 0 to about 8, or more, attached drug moieties. In addition, within each subgroup of conjugates with a particular integer ratio of drug moieties to a single antibody, there is a potentially heterogeneous mixture where the drug moiety is attached at various sites on the antibody. Analytical and preparative methods are inadequate to separate and characterize the antibody-drug conjugate species molecules within the heterogeneous mixture resulting from a conjugation reaction. Antibodies are large, complex and structurally diverse biomolecules, often with many reactive functional groups. Antibody reactivity with linker reagents and drug-linker intermediates are dependent on factors such as pH, concentration, salt concentration, and co-solvents. Furthermore, the multistep conjugation process may be nonreproducible due to difficulties in controlling the reaction conditions and characterizing reactants and intermediates.
Antibody-drug conjugates are typically formed by conjugating one or more antibody cysteine thiol groups to one or more linker moieties bound to a drug thereby forming an antibody-linker-drug complex. Cysteine thiols are reactive at neutral pH, unlike most amines which are protonated and less nucleophilic near pH 7. Since free thiol (RSH, sulfhydryl) groups are relatively reactive, proteins with cysteine residues often exist in their oxidized form as disulfide-linked oligomers or have internally bridged disulfide groups. Antibody cysteine thiol groups are generally more reactive, i.e. more nucleophilic, towards electrophilic conjugation reagents than antibody amine or hydroxyl groups. Engineering in cysteine thiol groups by the mutation of various amino acid residues of a protein to cysteine amino acids is potentially problematic, particularly in the case of unpaired (free Cys) residues or those which are relatively accessible for reaction or oxidation. In concentrated solutions of the protein, whether in the periplasm of E. coli, culture supernatants, or partially or completely purified protein, unpaired Cys residues on the surface of the protein can pair and oxidize to form intermolecular disulfides, and hence protein dimers or multimers. Disulfide dimer formation renders the new Cys unreactive for conjugation to a drug, ligand, or other label. Furthermore, if the protein oxidatively forms an intramolecular disulfide bond between the newly engineered Cys and an existing Cys residue, both Cys groups are unavailable for active site participation and interactions. Furthermore, the protein may be rendered inactive or non-specific, by misfolding or loss of tertiary structure (Zhang et al. (2002) Anal. Biochem. 311:1-9).
Improved antibody-drug conjugates, THIOMAB™, have been developed that provide for site-specific conjugation of a drug to an antibody through cysteine substitutions at sites where the engineered cysteines are available for conjugation but do not perturb immunoglobulin folding and assembly or alter antigen binding and effector functions (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485; WO2009/052249). These THIOMAB™ antibodies can then be conjugated to cytotoxic drugs through the engineered cysteine thiol groups to obtain THIOMAB™ drug conjugates (TDC) with uniform stoichiometry (e.g., up to 2 drugs per antibody in an antibody that has a single engineered cysteine site). Studies with multiple antibodies against different antigens have shown that TDCs are as efficacious as conventional antibody-drug conjugate in xenograft models and are tolerated at higher doses in relevant preclinical models. THIOMAB™ antibodies have been engineered for drug attachment at different locations of the antibody (e.g., specific amino acid positions (i.e., sites) within the light chain-Fab, heavy chain-Fab and heavy chain-Fc). The in vitro and in vivo stability, efficacy and PK properties of THIOMAB™ antibodies provide a unique advantage over conventional antibody-drug conjugates due to their homogeneity and site-specific conjugation to cytotoxic drugs.
There are still other limitations or challenges to the preparation and use of antibody-drug conjugates, and in particular antibody-calicheamicin derivative conjugates. For example, some linkers may be labile in the blood stream, thereby releasing unacceptable amounts of the drug prior to internalization in a target cell. Other linkers may provide stability in the bloodstream, but intracellular release effectiveness may be negatively impacted. Linkers that provide for desired intracellular release typically have poor stability in the bloodstream. Alternatively stated, bloodstream stability and intracellular release are typically inversely related. Second, in standard conjugation processes, the amount of calicheamicin loaded on the carrier protein (the drug loading), the amount of aggregate that is formed in the conjugation reaction, and the yield of final purified conjugate that can be obtained are interrelated. For example, aggregate formation is generally positively correlated to the number of equivalents of calicheamicin and derivatives thereof conjugated to the carrier-antibody. Because drug potency and efficacy increases with calicheamicin content, it is desirable to maximize calicheamicin loading on an antibody carrier while retaining the affinity of the antibody. However, under high drug loading, formed aggregates must be removed for therapeutic applications. As a result, drug loading-mediated aggregate formation decreases antibody-drug conjugate yield and can renders process scale-up difficult. For example, prior art conjugation methods using linkers have been found to require a compromise between higher drug loading and antibody-drug conjugate yield, by limiting the amount of calicheamicin that is added to the conjugation reaction.
Accordingly, there is a continuing need for improved efficacious calicheamicin-antibody conjugates that provide for optimized safety and efficacy.